Is There a Role for Tedizolid in the Treatment of Non-Tuberculous Mycobacterial Disease?

J Antimicrob Chemother 2020; 75: 609–617 doi:10.1093/jac/dkz511 Advance Access publication 30 December 2019

Is there a role for tedizolid in the treatment of non-tuberculous mycobacterial disease?

Mike Marvin Ruth 1, Valerie A. C. M. Koeken2, Lian J. Pennings1, Elin M. Svensson3,4, Heiman F. L. Wertheim1, Wouter Hoefsloot5 and Jakko van Ingen 1*

1Radboudumc Center for Infectious Diseases, Department of Medical Microbiology, Radboud University Medical Center, Nijmegen, Downloaded from https://academic.oup.com/jac/article-abstract/75/3/609/5690335 by guest on 06 April 2020 The Netherlands; 2Radboudumc Center for Infectious Diseases, Department of Internal Medicine, Radboud University Medical Center, Nijmegen, The Netherlands; 3Radboudumc Center for Infectious Diseases, Department of Pharmacy, Radboud University Medical Center, Nijmegen, The Netherlands; 4Department of Pharmaceutical Biosciences, Uppsala University, Uppsala, Sweden; 5Radboudumc Center for Infectious Diseases, Department of Pulmonary Diseases, Radboud University Medical Center, Nijmegen, The Netherlands

*Corresponding author. E-mail: [email protected]

Received 26 September 2019; returned 1 November 2019; revised 6 November 2019; accepted 8 November 2019

Background: Pulmonary infections caused by non-tuberculous mycobacteria (NTM) are hard to treat and have low cure rates despite intensive multidrug therapy. Objectives: To assess the feasibility of tedizolid, a new oxazolidinone, for the treatment of Mycobacterium avium and Mycobacterium abscessus. Methods: We determined MICs of tedizolid for 113 isolates of NTM. Synergy with key antimycobacterial drugs was assessed using the chequerboard method and calculation of the FIC index (FICI). We performed time–kill kinetics assays of tedizolid alone and combined with amikacin for M. abscessus and with ethambutol for M. avium. Human macrophages were infected with M. abscessus and M. avium and subsequently treated with tedizolid; intracellular and extracellular cfu were quantiﬁed over time. Results: NTM isolates generally had a lower MIC of tedizolid than of linezolid. FICIs were lowest between tedizolid and amikacin for M. abscessus (FICI = 0.75) and between tedizolid and ethambutol for M. avium (FICI = 0.72). Clarithromycin and tedizolid showed initial synergy, which was abrogated by erm(41)-induced macrolide resistance (FICI = 0.53). Tedizolid had a weak bacteriostatic effect on M. abscessus and combination with amikacin slightly prolonged its effect. Tedizolid had concentration-dependent activity against M. avium and its efﬁcacy was enhanced by ethambutol. Both combinations had a concentration-dependent synergistic effect. Tedizolid could inhibit the intracellular bacterial population of both M. avium and M. abscessus. Conclusions: Tedizolid should be further investigated in pharmacodynamic studies and clinical trials for M. avium complex pulmonary disease. It is less active against M. abscessus, but still promising.

Introduction addition, this multidrug treatment is associated with significant adverse effects.6 Non-tuberculous mycobacteria (NTM) are causative agents of The oxazolidinone linezolid is recommended as part of first-line chronic, opportunistic pulmonary infections in susceptible patient treatment of M. abscessus disease and as a second-line treatment populations.1 In NTM pulmonary disease (NTM-PD) Mycobacterium of MAC pulmonary disease.1 Because of the necessity of prolonged avium complex (MAC) and Mycobacterium abscessus are the most treatment, the linezolid dose has to be lowered to avoid or delay common causative pathogens.2,3 Current treatment regimens adverse events.7 Given linezolid’s very high MICs (median 16 mg/L),8 consist of at least three drugs for a duration of 18–24 months,1,4 these low doses are unlikely to be effective. but treatment outcome is still poor. In meta-analyses, 50% Tedizolid is an oxazolidinone that, like linezolid, targets the 23S (M. abscessus) to 70% (MAC) of patients who tolerate recom- ribosomal RNA of the 50S ribosomal subunit.9 Its favourable tox- mended regimens achieve prolonged culture conversion.5 In icity profile and superior penetration into epithelial lining fluid,

VC The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 609 Ruth et al.

compared with linezolid, has already led to sporadic use in NTM-PD bottles containing 10 mL of CAMHB with 0.05% Tween 80 and for M. avium treatment.10–12 A recent study using an intracellular hollow-fibre 10% OADC growth supplement (BD Biosciences). We tested tedizolid system infection model identified tedizolid as highly bactericidal concentrations ranging from 0.25% to 32% MIC and a growth control, all 5 6 13 inoculated with a bacterial inoculum approximating 10 –10 cfu/mL and against intracellular MAC, but lower in vitro activity of tedizolid alone against M. abscessus is reported.8,14,15 incubated at 30 C (for M. abscessus)or37 C(M. avium), shaken constantly and ventilated through a filter. We used the following predetermined time- Even with promising in vitro performance and toxicity profile, points: days 0, 1, 2, 3, 4, 5, 7, 14 and 21 for M. abscessus and an additional any new drug to be incorporated into NTM disease treatment day 28 for M. avium. At these timepoints, the bacterial population of each regimens should have a firm preclinical basis, including at least bottle was quantified using a 10-fold serial dilution with triplicate spot- measurements of synergy with established antimycobacterial plating of each well on M7H10 agar plates. Plates were incubated at 30C drugs and intracellular activity. for 3 days for M. abscessus and at 37C for 7 days for M. avium. Time–kill kin-

Here, we assess the in vitro and ex vivo activity of tedizolid as well etics for tedizolid interactions were determined following the protocol Downloaded from https://academic.oup.com/jac/article-abstract/75/3/609/5690335 by guest on 06 April 2020 as synergism between tedizolid and key antimycobacterial drugs described above. Single-drug tedizolid, companion and a tedizolid/compan- using the chequerboard method and time–kill kinetics assays. ion combination were tested with three concentrations (0.5%,1% and 2% MIC) in CAMHB. We defined a bactericidal effect as a >1 log cfu decrease in Methods the course of the interaction compared with the start inoculum. To assess the resistant M. avium and M. abscessus populations in the combination Bacterial strains time–kill kinetics assays, we prepared Middlebrook 7H11 (M7H11) agar plates containing 5% MIC of the companion drug and, to assess some M. abscessus subspecies abscessus CIP 104536, Mycobacterium fortuitum form of oxazolidinone resistance, 5% MIC of linezolid as an indicator for ATCC 6841, M. avium ATCC 700898, Mycobacterium peregrinum ATCC measuring emerging tedizolid resistance. M7H11 plates were prepared 700686, Mycobacterium chimaera DSM 44623, Mycobacterium simiae ATCC from powder (Sigma–Aldrich) according to the manufacturer’s recommen- 25275 and Mycobacterium intracellulare DSM 43223 reference strains were dation, with 10% OADC growth supplement and antibiotic added after purchased from their relevant culture collections. Clinical isolates were autoclaving. Plates were inoculated as described before. Inoculated M7H11 acquired from the collection of the Department of Medical Microbiology at plates were kept for 10 days at 30CforM. abscessus and for 21 days at Radboud University Medical Center, Nijmegen, The Netherlands. Isolates 37CforM. avium. were stored in trypticase soy broth with 40% glycerol at #80C and freshly cultured before each experiment. Response surface analysis Susceptibility and stability testing Response surface analysis to assess interaction according to Bliss independ- MICs of tedizolid phosphate (the active moiety of tedizolid; henceforth ence was performed as previously described.20 AUC was calculated from referred to as tedizolid; MSD) were determined by broth microdilution in the log cfu versus time plots using the trapezoidal rule after averaging the CAMHB (BD Biosciences, Drachten, the Netherlands) according to CLSI result form the two replicates and normalizing to the baseline colony count. guidelines.16 We tested concentrations ranging from 0.125 to 128 mg/L on The effect was then calculated according to effectx =AUCgrowth control # AUCx, a total of 113 NTM isolates (7 reference strains and 106 clinical isolates). where x is any given curve other than the growth control. MBCs were determined for reference strains of M. abscessus and M. avium To assess potential interactions in the time–kill kinetics experiments we by quantification of cfu for tedizolid concentrations that showed no visible calculated the expected effect for a combination under Bliss independence growth in microdilution, on Middlebrook 7H10 agar plates (BD Biosciences). (Ecomb,BI) to be compared with the observed effect (Ecomb,obs). Since Bliss in- To determine whether tedizolid should be considered bacteriostatic or bac- dependence builds on probability theory, the maximum effect to be eval- tericidal, we calculated the MBC/MIC ratio and considered a ratio >8 to be 17 uated is limited to 1 and therefore all effects were normalized to the Emax bacteriostatic, as previously described. We tested tedizolid stability by of the most potent drug (tedizolid). After simplification this gives the follow- pre-incubating broth microdilution assay plates for 7, 14 and 21 days at ing formula: Ecomb,BI = EA ! EB # (EA % EB)/Emaxhigh,whereEA and EB are the 30Cor37C before inoculating them with M. abscessus CIP 104536 or M. effect sizes of drug A or B separately and Emaxhigh is the highest maximum avium ATCC 700898, respectively. To assess a correlation between linezolid effect. The Emax of tedizolid was determined by fitting a sigmoidal Emax ! and tedizolid MIC, we ln(x 1) transformed the MIC values and performed model to the concentration–response data using ordinary least squares. aSpearman’sq two-tailed correlation test using a Gaussian approximation. The difference between observed and expected effect (DE)was quantified as a percentage difference relative to the expected: Synergy testing DE =(Ecomb,obs # Ecomb,BI)/Ecomb,BI.WedefinedaDE of 0±10% as no inter- We assessed synergy between tedizolid and other antimycobacterial drugs action, anything less than #10% was defined as antagonistic and anything using the broth microdilution chequerboard method.18 Tedizolid was eval- more than 10% was defined as synergistic. uated against a total of 12 clinical and reference strains of NTM [tedizolid in combination with clarithromycin, cefoxitin, tigecycline or amikacin for rapidly growing mycobacteria (RGM; M. abscessus, M. fortuitum, M. peregrinum Ex vivo intracellular assays and M. chelonae) and tedizolid in combination with clarithromycin, rifampi- PBMCs were isolated from buffy coats obtained from three healthy volun- cin, ethambutol, amikacin or minocycline for M. avium]aspreviously 19 teers (Sanquin Bloodbank, Nijmegen, The Netherlands). Informed consent described. Drug interactions were interpreted according to the FIC index from these volunteers was obtained for use of their blood for scientific (FICI) and were classified as synergistic (FICI 0.5), having no interaction purposes, as approved by the Ethics Committee of Radboud University (FICI >0.5–4.0) or antagonistic (FICI >4.0). We selected the most promising Medical Center, Nijmegen, The Netherlands. Macrophage differentiation candidate antibiotics based on the FICI value for subsequent experiments was performed as described previously.21 One day prior to the experiment, in time–kill interaction assays. cells were dissociated and re-seeded in antibiotic-free medium in a 96-well plate (1%105 cells/well). Time–kill kinetics assays On the day of infection, the cell culture medium was removed and Time–kill kinetics assays were performed with M. abscessus and M. avium M. avium was added to the macrophages at an moi of 1:5 (cells:bacteria), reference strains in duplicate. In short, bacteria were cultured in individual and M. abscessus was added at an moi of 1:1. The macrophages were

610 Tedizolid against non-tuberculous mycobacteria JAC incubated with mycobacteria for 1 h at 37C to allow phagocytosis before but, after 14 days of pre-incubation, we measured increasing tedi- medium was changed to RPMI supplemented with 10% human pooled zolid MICs as well as some sedimentation at higher concentrations serum. Tedizolid was added at 2% MIC and was present for the remainder (Table S1). of the experiment. At the indicated timepoints, the number of extracellular bacteria was quantiﬁed through plating of the supernatant on Middlebrook 7H10 agar for cfu counts. To determine the number of intracellular bacteria, Synergism of tedizolid with other drugs macrophages were washed with warm PBS and then subjected to hypoton- FICIs of tedizolid combinations are shown in Table 2.For ic lysis in sterile water for 10 min. The intracellular fraction was then also quantiﬁed through serial dilutions and plating of the different dilutions on M. abscessus, the tedizolid/amikacin combination yielded the low- Middlebrook 7H10 plates. All samples were analysed in triplicate. est FICI (average = 0.75; range = 0.56–1). Notably, the lowest FICI for tedizolid/amikacin was observed for the M. abscessus reference

strain (FICI = 0.56). Though the initial synergism between tedizolid Downloaded from https://academic.oup.com/jac/article-abstract/75/3/609/5690335 by guest on 06 April 2020 and clarithromycin seemed promising, it was rapidly abrogated by Statistics emergence of induced clarithromycin resistance facilitated by the Calculations were performed using GraphPad Prism version 5.03 (GraphPad erm(41) gene. From this, we chose amikacin as a combination drug Software Inc., La Jolla, CA, USA) or R version 3.1.2 (R Foundation for for subsequent time–kill assays against M. abscessus. A combination Statistical Computing, Vienna, Austria; https://www.R-project.org/). Error of tedizolid and cefoxitin was synergistic against M. fortuitum bars in the ﬁgures show SEMs. (FICI = 0.375) and a tedizolid/tigecycline combination was synergistic against M. peregrinum (FICI = 0.375). Results Tedizolid had no average synergistic interaction for MAC iso- MICs, MBCs and stability lates, but had isolated FICIs of 0.5 (Table 2). The lowest average FICI was found in combination with ethambutol (average = 0.72; The MICs of tedizolid for clinically relevant NTM are given in Table 1. range = 0.5–1). From this, we chose ethambutol as a combination MICs were generally lower for RGM than for slowly growing myco- drug for subsequent time–kill assays against M. avium.We bacteria (SGM). For both M. abscessus (Figure 1a and b) and observed no antagonistic interactions. M. avium (Figure 1c and d), there is a trend visible for more potent in vitro activity of tedizolid compared with linezolid against NTM. For M. abscessus, tedizolid and linezolid MICs are not correlated Time–kill kinetics assays (Spearman’s q = 0.2304, P =0.1192;Figure1e), but they are corre- The time–kill kinetics for M. abscessus are shown in Figure 2(a) for lated for M. avium (Spearman’s q =0.5921, P < 0.0001; Figure 1f). the tedizolid dose–response assay and Figure 2(b) for the compan- Individual linezolid and tedizolid MICs per isolate are given in Table S1 ion time–kill kinetics for the tedizolid/amikacin combination assay. (available as Supplementary data at JAC Online). All wells without vis- All conditions of tedizolid show growth until day 2, after which the ible growth still produced cfu after plating, regardless of organism, bacterial loads remain static until day 5 or continued a slightly leading to an MBC of >128 mg/L for both M. avium and M. abscessus inhibited growth following a concentration-dependent pattern. and MBC/MIC ratios of 16 for M. avium and 32–64 for M. abscessus, Notably, the 32% MIC combination shows the second highest bac- deﬁning tedizolid as bacteriostatic. Tedizolid MICs remained stable terial load by day 21. The combination time–kill kinetics assays in for M. abscessus and M. avium after a pre-incubation period of 7 days, Figure 2(b) show over the course of the experiment that the

Table 1. Tedizolid and linezolid MICs for clinical isolates of SGM and RGM, in CAMHB; for SGM, the broth was enriched with 10% OADC growth supplement

Organism n MIC50/MIC90 or MIC (mg/L) Tedizolid Linezolid

RGM

M. abscessus 47 MIC50/MIC90 2/8 8/>32 M. abscessus CIP 104536 MIC 4 >32 M. fortuitum 2 MIC 0.25 2 M. fortuitum ATCC 6841 MIC 2 8 M. peregrinum ATCC 700686 MIC 1 NA Mycobacterium chelonae 2 MIC 0.5–1 8–16 SGM

M. avium 51 MIC50/MIC90 2/16 16/32 M. avium ATCC 700898 MIC 8 16 M. chimaera 2 MIC 4 8–16 M. chimaera DSM 44623 MIC 1 4 M. intracellulare 2 MIC 4–8 16–32 M. intracellulare DSM 43223 MIC 2 <1 M. simiae ATCC 25275 MIC 2 16

NA, not applicable.

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(a)M. abscessus - LZD (b) M. abscessus - TZD 25 1.0 15 1.0

20 0.8 0.8 10 15 0.6 0.6

10 0.4 0.4

5 Cumulative proportion of isolates 5 0.2 0.2 Downloaded from https://academic.oup.com/jac/article-abstract/75/3/609/5690335 by guest on 06 April 2020 0 0 0 0 2 4816 32 >32 0.25 0.5 1 2 4 8 16 M. avium - LZD M. avium - TZD (c) (d)

20 1.0 15 1.0 Number of isolates

0.8 0.8 15 10 0.6 0.6 10 0.4 0.4 5 5 0.2 0.2

0 0 0 0 248163264 0.25 0.5 1 2 4 8 16 32 64

MIC in mg/L

(e)M. abscessus (f) M. avium 3 5.0

2 2.5

1 TZD MIC (In+1) TZD MIC (In+1)

1.10 1.61 2.19 2.83 3.49 4.17 1.10 1.61 2.19 2.83 3.49 4.17 LZD MIC (In+1) LZD MIC (In+1)

Figure 1. MIC distributions of linezolid and tedizolid for clinical isolates of M. abscessus (n = 47) and M. avium (n = 51). (a) Linezolid MIC distribution for M. abscessus. (b) Tedizolid MIC distribution for M. abscessus. (c) Linezolid MIC distribution for M. avium. (d) Tedizolid MIC distribution for M. avium. (e) Correlation between linezolid MIC and tedizolid MIC for M. abscessus. MICs were not correlated (n = 47, Spearman’s q = 0.2304, two tailed, P = 0.1192). (f) Correlation between linezolid MIC and tedizolid MIC for M. avium. MICs are correlated (n = 51, Spearman’s q = 0.5921, two tailed, P < 0.0001). LZD, linezolid; TZD, tedizolid. combination conditions have a consistently lower bacterial burden shows a more pronounced bacteriostatic effect, but all conditions than the single-drug conditions and the 2% MIC combination con- show sustained growth by day 10. The percentage of the dition has a static bacterial burden until day 10. An exception to amikacin-resistant population is shown in Figure 2(c). All treatment this overall trend is the 0.5% MIC combination condition, which conditions induced amikacin resistance, but conditions treated does not seem to prolong the bacteriostatic activity compared with amikacin showed the highest resistant subpopulation. The with the single-drug conditions. Compared with tedizolid, amikacin percentage of the linezolid-resistant subpopulation as a proxy for

612 Tedizolid against non-tuberculous mycobacteria JAC

Table 2. FICIs of tedizolid in combination with clarithromycin, cefoxitin, tigecycline or amikacin for RGM, and tedizolid in combination with clarithromycin, rifampicin, ethambutol, amikacin or minocycline for M. avium

M. abscessus (n =5) M. fortuitum (n =1) M. peregrinum (n =1) M. chelonae (n =1) Combination average FICI range (min–max) FICI FICI FICI

Tedizolid/clarithromycin 0.53 0.28–0.625 0.75 1 1 Tedizolid/cefoxitin 0.81 0.56–1 0.375 0.625 0.5 Tedizolid/tigecycline 1.01 0.56–1.25 1 0.375 0.625 Tedizolid/amikacin 0.75 0.56–1 0.75 0.5 0.75 Downloaded from https://academic.oup.com/jac/article-abstract/75/3/609/5690335 by guest on 06 April 2020 MAC (n =4)

Combination average FICI range (min–max)

Tedizolid/clarithromycin 1 0.75–1.25 Tedizolid/rifampicin 1.25 0.5–2.5 Tedizolid/ethambutol 0.72 0.5–1 Tedizolid/amikacin 0.81 0.5–1 Tedizolid/minocycline 0.78 0.625–1

the tedizolid-resistant subpopulation is shown in Figure 2(d). No tedizolid and ethambutol is considered synergistic at lower con- clear trends are visible and all conditions induce linezolid resist- centrations (DE = 42.3%) and turns slightly antagonistic at higher ance similarly. concentrations (DE = #13.4%). The time–kill kinetics for M. avium are shown in Figure 3(a) for the tedizolid dose–response assay and Figure 3(b) for the companion time–kill kinetics for the tedizolid/ethambutol combination assay. At Ex vivo intracellular assays concentrations higher than 1% MIC, tedizolid achieves at least Intracellular and extracellular cfu/mL counts of M. avium are >1 log kill by day 28. Notably, the 4% MIC condition does not follow shown in Figure 4(a). Tedizolid reduced the intracellular bacterial the same dose–response trend and has a higher bacterial burden on load by 66% below stasis over the course of 6 days. The extracellu- day 28 than the 2% MIC condition. The combination time–kill kinet- lar bacterial population was comparatively low (2% of total cfu ics assays in Figure 3(b) show that, overall, the combination condi- counts). Intracellular and extracellular cfu/mL counts of tions have a lower bacterial burden than the single-drug conditions M. abscessus are shown in Figure 4(b). Overall, tedizolid could hold and all conditions follow a dose–response distribution. In most com- the bacterial load static compared with the non-treated control bination conditions, we observe that the SEM exceeds 1 log at some over the course of 3 days. Especially on day 2, we observe a large point, indicating high variability between samples. Both the 1% MIC extracellular M. abscessus population in the non-treated controls, and 2% MIC combination conditions end on a par with the 2% MIC but not in the treated conditions. By day 3, the intracellular bacter- tedizolid condition on day 28. We did not detect a linezolid-resistant ial load of the treated conditions is lower by a factor of 500 than subpopulation in any of the conditions (data not shown). the non-treated condition. Notably, little extracellular bacterial load is detected in the tedizolid-treated conditions. Response surface analysis Discussion The sigmoidal Emax curves for tedizolid on M. abscessus and M. avium are shown in Figure S1(a) and Figure S1(b), respectively. Tedizolid holds promise as the successor to linezolid in the treatment 8 For M. abscessus, the fitted Emax was 34.1 log10 cfu/mL % day, of mycobacterial diseases, because of its increased in vitro activity the EC50 was 0.73% MIC and the estimated Hill slope was 2.63. For and lower toxicity in treatment of other bacterial infections, com- 12,22 M. avium, the fitted Emax was 176 log10 cfu/mL % day, the EC50 paredwithlinezolid. Linezolid is recommended in M. abscessus was 0.94% MIC and the estimated Hill slope was 0.84. The calcu- therapy and is a second-line agent in MAC disease treatment,1 lated DE percentages as well as Ecomb,obs, Ecomb,BI and the effect although there are no clinical data supporting its efficacy. A more ac- sizes of the companion drugs in the combination time–kill kinetics tive and better-tolerated alternative to linezolid is desirable. In this assays are shown in Table 3. study, we confirm previous MIC distribution data for MAC and The combination of tedizolid and amikacin against M. abscessus found by Brown-Elliott and Wallace8 and also find that M. abscessus had an antagonistic interaction at low concentrations tedizolid generally has a lower MIC than linezolid. Notably, the (DE = #126%) and was synergistic at higher concentrations tedizolid MIC distribution for M. avium seems to be bimodal, empha- (DE = 39.7%). This is mainly because of a prolonged lower cfu count sizing the need to perform in vitro drug susceptibility testing. The from day 6 onwards, after which the 2% MIC tedizolid/amikacin mechanistic background underlying this bimodal MIC distribution condition performed better than the single-drug conditions as requires further study. Tedizolid showed concentration-dependent it prevented regrowth. For M. avium, the interaction between activity against M. avium and bacteriostatic activity against

613 Ruth et al.

(a) (b) 10 10

8 × cfu/mL 8 cfu/mL 2 MIC TZD 10 32 × MIC 10 1 × MIC TZD × × log 16 MIC log 0.5 MIC TZD 8 × MIC 2 × MIC TZD/AMK 4 × MIC 1 × MIC TZD/AMK 6 × × Downloaded from https://academic.oup.com/jac/article-abstract/75/3/609/5690335 by guest on 06 April 2020 6 2 MIC 0.5 MIC TZD/AMK 1 × MIC 2 × MIC AMK M. abscesus M. abscesus 0.5 × MIC 1 × MIC AMK 0.25 × MIC 0.5 × MIC AMK Growth control 4 Growth control 051015 20 051015 20 Days Days

1000 1000 100 10 100 Growth control × 1 Growth control 2 MIC TZD 2 × MIC TZD 1 × MIC TZD 0.1 1 × MIC TZD MIC LZD-resistant cfu 0.5 × MIC TZD × 10 MIC AMK-resistant cfu 0.5 × MIC TZD

× × × 2 MIC TZD/AMK 0.01 2 MIC TZD/AMK 1 × MIC TZD/AMK 1 × MIC TZD/AMK × 0.5 × MIC TZD/AMK 0.001 0.5 MIC TZD/AMK 1 × 2 × MIC AMK 2 MIC AMK 0.0001 1 × MIC AMK 1 × MIC AMK 0.5 × MIC AMK Percentage 5 0.5 × MIC AMK Percentage 5 0.00001 100% 0.1 100% 51015 20 51015 20 Days Days

Figure 2. Time–kill kinetics of tedizolid against M. abscessus CIP 104536. (a) Dose–response of tedizolid (MIC = 4 mg/L). (b) Combination time–kill kinetics of tedizolid and amikacin (MIC = 32 mg/L). (c) Percentage of the 5% MIC amikacin-resistant subpopulation of (b). (d) Percentage of the 5% MIC linezolid-resistant subpopulation of (b). AMK, amikacin; LZD, linezolid; TZD, tedizolid.

(a) (b)

8 8

2 × MIC TZD 6 32 × MIC 6 1 × MIC TZD cfu/mL 16 × MIC

10 × cfu/mL 0.5 MIC TZD × 8 MIC 10 ×

log 2 MIC TZD/EMB 4 4 × MIC log 4 1 × MIC TZD/EMB × 2 MIC 0.5 × MIC TZD/EMB × 1 MIC 2 × MIC EMB M. avium 2 × × 0.5 MIC M. avium 2 1 MIC EMB 0.25 × MIC 0.5 × MIC EMB Growth control Growth control 0 Detection limit 0 Detection limit 0102030 0102030 Days Days

Figure 3. Time–kill kinetics of tedizolid against M. avium ATCC 700898. (a) Dose–response of tedizolid (MIC = 8 mg/L). (b) Combination time–kill kinetics of tedizolid and ethambutol (MIC = 4 mg/L). EMB, ethambutol; TZD, tedizolid.

614 Tedizolid against non-tuberculous mycobacteria JAC

Table 3. Response surface analysis results, as well as interaction effect size, by deviation from expected combination effect under Bliss independence in percentage DE

Effect (log10 cfu/mL % day)

Combination (concentration) tedizolid amikacin or ethambutol observed, combination expected, combinationa DE (%)

M. abscessus tedizolid/amikacin (0.5% MIC) 8.93 #95.6 16.3 62.2 #126 tedizolid/amikacin (1% MIC) 22.6 24.2 33.7 31.1 8.24 tedizolid/amikacin (2% MIC) 34.8 35.6 48.8 34.9 39.7

M. avium Downloaded from https://academic.oup.com/jac/article-abstract/75/3/609/5690335 by guest on 06 April 2020 tedizolid/ethambutol (0.5% MIC) 65.8 9.39 102 71.6 42.3 tedizolid/ethambutol (1% MIC) 96.5 81.8 145 133 8.81 tedizolid/ethambutol (2% MIC) 137 95.1 137 159 #13.4 aUnder Bliss independence.

(a) (b) 107 Cell fraction Supernatant 106 Cell fraction Supernatant 106 105 cfu/mL

cfu/mL 104

105

M. avium 103 M. abscesscus

102

4 TZD TZD 10 TZD TZD TZD Inoculum Non-treated Non-treated Inoculum Non-treated Non-treated Non-treated

Day 0 Day 3 Day 6 Day 0 Day 1 Day 2 Day 3

Figure 4. Ex vivo infection assays of mycobacteria-infected macrophages treated with and without tedizolid (2% MIC). (a) Intracellular and extracellular cfu counts of M. avium. (b) Intracellular and extracellular cfu counts of M. abscessus. TZD, tedizolid.

M. abscessus in time–kill experiments and promising intracellular over 14 days, which translates well to our observed killing effect in activity against both M. avium and M. abscessus. time–kill assays. These researchers also assessed tedizolid to- With our time–kill experiments with M. abscessus,weconfirma gether with a ceftazidime/avibactam ! rifabutin ! moxifloxacin study performed by Compain et al.,14 who report modest exposure- combination in a hollow-fibre system infection model, reporting a dependent activity of tedizolid. Combination experiments with stronger killing effect than an azithromycin/ethambutol/rifabutin tedizolid and amikacin reveal modest, concentration-dependent regimen.23 Though these results are promising, adding tedizolid to synergy relative to Bliss independence. Tedizolid’s potential is further the standard regimen or exploiting our observed synergism with supported by its performance alone in human macrophages, where ethambutol by replacing rifampicin might be an approach more it holds the bacterial burden static over 3 days at 2% MIC. A previous translatable to clinical trials. study showed similar tedizolid activity against M. abscessus,in Translating in vitro findings to the clinical reality is challenging, THP-1 cells.15 especially when integrating tedizolid in a multidrug regimen For M. avium,fewin vitro studies beyond MIC determinations exploiting its synergism with ethambutol. Deshpande et al.13 pro- are available. The aforementioned hollow-fibre system study by pose a dose of 200 mg daily if no interacting co-medication is 13 Deshpande et al. shows intracellular killing of 2.0 log10 cfu/mL used, treating M. avium-infected THP-1 cells in a hollow-fibre

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system. Based on this study, Deshpande et al.13 suggested a tedizolid breakpoint of 1 mg/L. Based on our MIC distribution data, References a 1 mg/L breakpoint would define <40% of MAC isolates as 1 GriffithDE,AksamitT,Brown-ElliottBAet al. An official ATS/IDSA state- susceptible. ment: diagnosis, treatment, and prevention of nontuberculous mycobacterial A more favourable safety profile of tedizolid versus linezolid has diseases. Am J Respir Crit Care Med 2007; 175: 367–416. only been established for a 200 mg/day dose for up to 6 days;24 2 Hoefsloot W, van Ingen J, Andrejak C et al. The geographic diversity of non- the safety profile of prolonged administration of higher doses is tuberculous mycobacteria isolated from pulmonary samples: an NTM-NET uncertain. The efficacy and long-term safety of different tedizolid collaborative study. EurRespirJ2013; 42: 1604–13. doses within multidrug regimens should be investigated in phar- 3 Stout JE, Koh W-J, Yew WW. Update on pulmonary disease due to non- macodynamic models and ultimately clinical trials. tuberculous mycobacteria. Int J Infect Dis 2016; 45: 123–34.

Our study has several limitations to consider. Most importantly, 4 Floto RA, Olivier KN, Saiman L et al. US Cystic Fibrosis Foundation and Downloaded from https://academic.oup.com/jac/article-abstract/75/3/609/5690335 by guest on 06 April 2020 all our assays are static assays where drug is added only at the be- European Cystic Fibrosis Society consensus recommendations for the man- ginning and not continuously, making human pharmacokinetics agement of non-tuberculous mycobacteria in individuals with cystic fibrosis: impossible to model. A hollow-fibre system experiment might be executive summary. Thorax 2016; 71: 88–90. interesting, especially if assessing the tedizolid/ethambutol syner- 5 van Ingen J, Ferro BE, Hoefsloot W et al. Drug treatment of pulmonary gism as well as counteracting drug stability issues and medium nontuberculous mycobacterial disease in HIV-negative patients: the evi- dence. Expert Rev Anti Infect Ther 2013; 11: 1065–77. exhaustion. The high proportion of resistant cfu in the resistance monitoring assays surpassing 100% remains unexplained. 6 Philley JV, Griffith DE. Management of nontuberculous mycobacterial (NTM) lung disease. Semin Respir Crit Care Med 2013; 34: 135–42. Regrettably, we could also not monitor the linezolid resistance in M. avium time–kill assays. For this, lower concentrations of linezolid 7 Winthrop KL, Ku JH, Marras TK et al. The tolerability of linezolid in the treatment of nontuberculous mycobacterial disease. EurRespirJ2015; 45: (3% MIC) might have been helpful. Also, linezolid was merely used 1177–9. as a proxy for tedizolid resistance and it might not mirror the actual 8 Brown-Elliott BA, Wallace RJ. In vitro susceptibility testing of tedizolid resistance to tedizolid in M. abscessus resistance monitoring, pos- against nontuberculous mycobacteria. JClinMicrobiol2017; 55: sibly leading to an overestimation of resistance in these assays. 1747–54. Lastly, translating MICs, synergy and time–kill kinetics to possible 9 Burdette SD, Trotman R. Tedizolid: the first once-daily oxazolidinone class clinical efficacy is challenging, especially for M. fortuitum and antibiotic. Clin Infect Dis 2015; 61: 1315–21. M. peregrinum, where we only tested the reference strains. Ideally, 10 Conte JE Jr, Golden JA, Kipps J et al. Intrapulmonary pharmacokinetics of this will be established in a stepwise process of hollow-fibre system linezolid. Antimicrob Agents Chemother 2002; 46: 1475–80. studies and clinical trials. 11 Housman ST, Pope JS, Russomanno J et al. Pulmonary disposition of tedi- Concluding, we confirmed tedizolid as a potent asset in MAC zolid following administration of once-daily oral 200-milligram tedizolid pulmonary disease treatment; it is less active against M. abscessus, phosphate in healthy adult volunteers. Antimicrob Agents Chemother 2012; but still interesting as a replacement for linezolid. With the 56: 2627–34. observed synergistic interactions, especially with ethambutol, a 12 Yuste JR, Berto´J,DelPozoJLet al. Prolonged use of tedizolid in a tedizolid/ethambutol/azithromycin regimen might be an interest- pulmonary non-tuberculous mycobacterial infection after linezolid-induced ing candidate regimen for further pharmacodynamic studies and toxicity. J Antimicrob Chemother 2017; 72:625–8. ultimately clinical trials in MAC pulmonary disease. 13 Deshpande D, Srivastava S, Pasipanodya JG et al. Tedizolid is highly bactericidal in the treatment of pulmonary Mycobacterium avium complex disease. J Antimicrob Chemother 2017; 72 Suppl 2: ii30–5. 14 Compain F, Soroka D, Heym B et al. In vitro activity of tedizolid against Acknowledgements the Mycobacterium abscessus complex. Diagn Microbiol Infect Dis 2018; 90: We thank MSD for kindly providing us with tedizolid phosphate and ap- 186–9. preciate the collaboration. 15 Le Run E, Arthur M, Mainardi J-L. In vitro and intracellular activity of imipenem combined with tedizolid, rifabutin, and avibactam against Mycobacterium abscessus. Antimicrob Agents Chemother 2019; 63: Funding e01915-18. Jakko van Ingen is supported by a personal grant from the Netherlands 16 CLSI. Susceptibility Testing of Mycobacteria, Nocardiae, and Other Aerobic Organization for Scientific Research (NWO/ZonMW grant Veni Actinomycetes—Second Edition: M24. 2011. 016.176.024). This grant funded laboratory equipment as well as con- 17 Ruth MM, Sangen JJN, Pennings LJ et al. Minocycline has no clear role in sumables used in this study. the treatment of Mycobacterium abscessus disease. Antimicrob Agents Chemother 2018; 62: e01208-18. 18 Odds FC. Synergy, antagonism, and what the chequerboard puts be- Transparency declarations tween them. JAntimicrobChemother2003; 52:1. None to declare. 19 Ruth MM, Sangen JJN, Remmers K et al. A bedaquiline/clofazimine combination regimen might add activity to the treatment of clinically relevant non-tuberculous mycobacteria. J Antimicrob Chemother 2019; Supplementary data 74: 935–43. 20 Wicha SG, Kees MG, Kuss J et al. Pharmacodynamic and response surface Table S1 and Figure S1 are available as Supplementary data at JAC analysis of linezolid or vancomycin combined with meropenem against Online. Staphylococcus aureus. Pharm Res 2015; 32: 2410–8.

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21 Ruth MM, Magombedze G, Gumbo T et al. Minocycline treatment for pul- 23 Deshpande D, Srivastava S, Pasipanodya JG et al. A novel ceftazidime/avi- monary Mycobacterium avium complex disease based on pharmacokinetics/ bactam, rifabutin, tedizolid and moxiﬂoxacin (CARTM) regimen for pulmonary pharmacodynamics and Bayesian framework mathematical models. Mycobacterium avium disease. J Antimicrob Chemother 2017; 72 Suppl 2: JAntimicrobChemother2019; 74: 1952–61. ii48–53. 22 HallRG,SmithWJ,PutnamWCet al. An evaluation of tedizolid for 24 Lan S-H, Lin W-T, Chang S-P et al. Tedizolid versus linezolid for the treat- the treatment of MRSA infections. Expert Opin Pharmacother 2018; 19: ment of acute bacterial skin and skin structure infection: a systematic review 1489–94. and meta-analysis. Antibiotics (Basel) 2019; 8: E137. Downloaded from https://academic.oup.com/jac/article-abstract/75/3/609/5690335 by guest on 06 April 2020

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